The Endosomal Sorting Complex Required for Transport (ESCRT) is a membrane scission machine that functions to direct membrane budding away from the cytoplasm. Its role has been documented in several biological processes that are important to cellular homeostasis and defense against aging, including multi-vesicular body (MVB) biogenesis, membrane abscission during cytokinesis and autophagosome formation in autophagy. In addition, viruses such as HIV-1 and Ebola virus have hijacked the ESCRT machinery for their own usage in budding from the plasma membrane of infected human cells. Therefore, a detailed understanding of the molecular mechanism of the ESCRT function can provide insights into the pathophysiology of a range of human diseases from cancer, viral infection to neurodegeneration. The ESCRT machinery consists of four core ESCRT complexes (-0, -I, -II, and -III) and the Vps4 ATPase complex. It has been shown that the central reaction that drives membrane scission is executed by the ESCRT-III complex and fueled by Vps4. As the only energy-consuming enzyme in the ESCRT machinery, Vps4 is an excellent target for pharmacological intervention of the ESCRT function. A number of proteins including Vta1, Vps60, Did2 and Ist1 are regulators of Vps4 activity in vivo. The long-term goal of the proposal is to delineate the molecular mechanism that underlies the regulation of the Vps4 ATPase complex so that novel strategies can be devised for preventive and therapeutic discovery. Using a combined approach of structural biology, biochemistry and cell biology, the current project will pursue three specific aims: (1) to determine the structural basis of regulation of Vps4 oligomerization by Vta1;(2) to elucidate the molecular mechanism of action by Vps60;(3) to investigate the structural basis and biological role of Ist1 and Did2 interactions. These studies will produce high-resolution structural information describing the molecular interactions involving Vps4 and its associated regulators. Combined with insights gained from the concurrent structure/function relationship study, these results will significantly advance our understanding of the molecular mechanism underlying the function and regulation of Vps4. PUBLIC HEALTH RELEVANCE: The Vps4 ATPase complex is involved in a myriad of physiological and pathological processes that have implications in a number of human diseases including cancer, viral infection and neurodegeneration. Understanding the molecular mechanism underlying the function and regulation of Vps4 will enable us to target the protein complex for preventive and therapeutic discovery, which may lead to novel treatments for these diseases that have affected millions of lives.